Important, clinically relevant insights have emerged from our basic studies of the trafficking and processing of EGF receptor (EGFR) ligands in the context of polarized epithelial cells. TGFa and amphiregulin (AR) are delivered preferentially to the basolateral surface where they are cleaved by TACE/ADAM-17. Mature soluble TGFa then is avidly taken up by basolaterally restricted EGFRs. We have identified and characterized two TGFa cytoplasmic tail-interacting proteins (Naked2 and MAGI-3) that regulate, respectively, the fidelity and efficiency of TGFa delivery to the basolateral surface. Naked2 associates with TGFa-containing exocytic vesicles as they emerge from the Golgi, and escorts these vesicles to the basolateral plasma membrane where they dock and fuse in a Naked2 myristoylation-dependent manner. Functional Naked2 appears to be required for the proper basolateral delivery of TGFa since TGFa is "trapped" in the cytoplasm of myristoylation-deficient Naked2-expressing cells. Full length Naked2 protein is downregulated in colorectal cancer where TGFa immunostaining is often cytoplasmic. Studies are proposed to further examine the function of Naked2 and MAGI-3, and to test the hypothesis that trafficking of TGFa in polarized epithelial cells correlates with the oncogenic potential of an epithelial cell in that proper basolateral trafficking of TGFa contributes to the maintenance of epithelial homeostasis whereas aberrant trafficking of TGFa is tumor-promoting. In contrast to TGFa, AR is delivered to the basolateral surface in a Naked2-independent manner. AR binds basolateral EGFRs but less avidly than TGFa. Exogenous AR, but not TGFa, disrupts epithelial junctional integrity leading to an epithelial to mesenchymal-like transition. We hypothesize that a consequence of impaired delivery of TGFa to the basolateral surface is that AR binds "unoccupied" basolateral EGFRs, perturbs epithelial polarity and predisposes the cell to tumor progression. Based on our success in identifying proteins that functionally interact with the cytoplasmic tail of TGFa, we propose to conduct a similar analysis with the cytoplasmic tail of AR. Overall, these studies will elucidate mechanisms underlying the delivery of TGFa and AR to the basolateral surface of polarized epithelial cells, enhance our understanding of the role of these two EGFR ligands in epithelial cell biology and lead to important insights into the pathogenesis of colorectal cancer and provide novel, more effective ways to diagnose and treat this disease.